The present invention relates to cysteine proteases derived from gram-positive microorganisms. The present invention provides nucleic acid and amino acid sequences of cysteine protease 1, 2 and 3 identified in Bacillus. The present invention also provides methods for the production of cysteine protease 1, 2 and 3 in host cells as well as the production of heterologous proteins in a host cell having a mutation or deletion of part or all of at least one of the cysteine proteases of the present invention.
Gram-positive microorganisms, such as members of the group Bacillus, have been used for large-scale industrial fermentation due, in part, to their ability to secrete their fermentation products into the culture media. In gram-positive bacteria, secreted proteins are exported across a cell membrane and a cell wall, and then are subsequently released into the external media usually maintaining their native conformation.
Various gram-positive microorganisms are known to secrete extracellular and/or intracellular protease at some stage in their life cycles. Many proteases are produced in large quantities for industrial purposes. A negative aspect of the presence of proteases in gram-positive organisms is their contribution to the overall degradation of secreted heterologous or foreign proteins.
The classification of proteases found in microorganisms is based on their catalytic mechanism which results in four groups: the serine proteases; metalloproteases; cysteine proteases; and aspartic proteases. These categories can be distinguished by their sensitivity to various inhibitors. For example, the serine proteases are inhibited by phenylmethylsulfonylfluoride (PMSF) and diisopropylfluorophosphate (DIFP); the metalloproteases by chelating agents; the cysteine enzymes by iodoacetamide and heavy metals and the aspartic proteases by pepstatin. The serine proteases have alkaline pH optima, the metalloproteases are optimally active around neutrality, and the cysteine and aspartic enzymes have acidic pH optima (Biotechnology Handbooks, Bacillus. vol. 2, edited by Harwood, 1989 Plenum Press, New York).
The activity of cysteine protease depends on a catalytic dyad of cysteine and histidine with the order differing among families. The best known family of cysteine proteases is that of papain having catalytic residues Cys-25 and His-159. Cysteine proteases of the papain family catalyze the hydrolysis of peptide, amide, ester, thiol ester and thiono ester bonds. Naturally occurring inhibitors of cysteine proteases of the papain family are those of the cystatin family (Methods in Enzymology, vol. 244, Academic Press, Inc. 1994).
The present invention relates to the unexpected and surprising discovery of three heretofore unknown or unrecognized cysteine proteases found in Bacillus subtilis, designated herein as CP1, CP2 and CP3, having the nucleic acid and amino acid as shown in FIGS. 1A-1B, FIGS. 5A-5B and 6A-6B, respectively. The present invention is based in part, upon the presence of the characteristic cysteine protease amino acid motif GXCWAF found in uncharacterised translated genomic nucleic acid sequences of Bacillus subtilis. The present invention is also based in part upon the structural relatedness that CP1 has with the cysteine protease papain specifically with respect to the location of the catalytic histidine/alanine and asparagine/serine residues and the structural relatedness that CP1 has with CP2 and CP3.
The present invention provides isolated polynucleotide and amino acid sequences for CP1, CP2 and CP3. Due to the degeneracy of the genetic code, the present invention encompasses any nucleic acid sequence that encodes the CP1, CP2 and CP3 amino acid sequence shown in the Figures.
The present invention encompasses amino acid variations of B.subtilis CP1, CP2 and CP3 amino acids disclosed herein that have proteolytic activity. B. subtilis CP1, CP2 and CP3, as well as proteolytically active amino acid variations thereof, have application in cleaning compositions. In one aspect of the present invention, CP1, CP2 or CP3 obtainable from a gram-positive microorganism is produced on an industrial fermentation scale in a microbial host expression system. In another aspect, isolated and purified recombinant CP1, CP2 or CP3 obtainable from a gram-positive microorganism is used in compositions of matter intended for cleaning purposes, such as detergents. Accordingly, the present invention provides a cleaning composition comprising at least one of CP1, CP2 and CP3 obtainable from a gram-positive microorganism. The cysteine protease may be used alone in the cleaning composition or in combination with other enzymes and/or mediators or enhancers.
The production of desired heterologous proteins or polypeptides in gram-positive microorganisms may be hindered by the presence of one or more proteases which degrade the produced heterologous protein or polypeptide. Therefore, the present invention also encompasses gram-positive microorganism having a mutation or deletion of part or all of the gene encoding CP1 and/or CP2 and/or CP3, which results in the inactivation of the CP1 and/or CP2 and/or CP3 proteolytic activity, either alone or in combination with deletions or mutations in other proteases, such as apr, npr, epr, mpr for example, or other proteases known to those of skill in the art. In one embodiment of the present invention, the gram-positive organism is a member of the genus Bacillus. In another embodiment, the Bacillus is Bacillus subtilis. 
In another aspect, the gram-positive microorganism host having one or more deletions or mutations in a cysteine protease of the present invention is further genetically engineered to produce a desired protein. In one embodiment of the present invention, the desired protein is heterologous to the gram-positive host cell. In another embodiment, the desired protein is homologous to the host cell. The present invention encompasses a gram-positive host cell having a deletion or interruption of the naturally occurring nucleic acid encoding the homologous protein, such as a protease, and having nucleic acid encoding the homologous protein or a variant thereof re-introduced in a recombinant form. In another embodiment, the host cell produces the homologous protein. Accordingly, the present invention also provides methods and expression systems for reducing degradation of heterologous or homologous proteins produced in gram-positive microorganisms comprising the steps of obtaining a Bacillus host cell comprising nucleic acid encoding said heterologous protein wherein said host cell contains a mutation or deletion in at least one of the genes encoding cysteine protease 1, cysteine protease 2 and cysteine protease 3; and growing said Bacillus host cell under conditions suitable for the expression of said heterologous protein. The gram-positive microorganism may be normally sporulating or non-sporulating.
The present invention provides methods for detecting gram positive microorganism homologs of B. subtilis CP1, CP2 and CP3 that comprises hybridizing part or all of the nucleic acid encoding B. subtilis CP1, CP2 and CP3 with nucleic acid derived from gram-positive organisms, either of genomic or cDNA origin.